X-ray and biochemical analysis of N370S mutant human acid β-glucosidase

Abstract

Gaucher disease is caused by mutations in the enzyme acid β-glucosidase (GCase), the most common of which is the substitution of serine for asparagine at residue 370 (N370S). To characterize the nature of this mutation, we expressed human N370S GCase in insect cells and compared the x-ray structure and biochemical properties of the purified protein with that of the recombinant human GCase (imiglucerase, Cerezyme®). The x-ray structure of N370S mutant acid β-glucosidase at acidic and neutral pH values indicates that the overall folding of the N370S mutant is identical to that of recombinant GCase. Subtle differences were observed in the conformation of a flexible loop at the active site and in the hydrogen bonding ability of aromatic residues on this loop with residue 370 and the catalytic residues Glu-235 and Glu-340. Circular dichroism spectroscopy showed a pH-dependent change in the environment of tryptophan residues in imiglucerase that is absent in N370S GCase. The mutant protein was catalytically deficient with reduced V(max) and increased K(m) values for the substrate p-nitrophenyl-β-D-glucopyranoside and reduced sensitivity to competitive inhibitors. N370S GCase was more stable to thermal denaturation and had an increased lysosomal half-life compared with imiglucerase following uptake into macrophages. The competitive inhibitor N-(n-nonyl)deoxynojirimycin increased lysosomal levels of both N370S and imiglucerase 2-3-fold by reducing lysosomal degradation. Overall, these data indicate that the N370S mutation results in a normally folded but less flexible protein with reduced catalytic activity compared with imiglucerase.

FIGURE 1.

Structure of N370S GCase at neutral and acidic pH values. a, N370S GCase at pH 7.1. b, N370S GCase at pH 5.4. Green, domain I; pink, domain II; light blue, domain III. Catalytic residues are shown as orange sticks. Ser-370 is shown as red sticks. c, overlay of pH 7.1 N370S GCase structure (cyan) with imiglucerase at neutral pH (2NT1, pink). d, overlay of pH 5.4 N370S GCase structure (green) with imiglucerase at acidic pH (3GXI, orange).

FIGURE 2.

Active site loop conformations of N370S GCase. N370S GCase structures are shown in shades of blue at pH 7.1, and shades of green at pH 5.4. Imiglucerase structures are shown in shades of pink at neutral pH (2NT1), and shades of yellow at acidic ph (3GXI). Two conformations are shown for each structure.

FIGURE 3.

Loop 1 conformations and H-bonds with active sites and helix 7. a, N370S GCase at pH 7.1 in shades of blue. b, N370S GCase at pH 5.4 in shades of green. c, imiglucerase at neutral pH (2NT1) in shades of pink. d, imiglucerase at acidic pH (3GXI) in shades of yellow. Two conformations are shown for each structure. H-bonds are shown as dotted lines.

FIGURE 4.

Solution structure comparison and the conformation of Tyr and Trp residues in GCase. a, CD spectra of imiglucerase at pH 7.1 and 5.4. b, CD spectra of N370S at pH 7.1 and 5.4. c, overlay of all Trp residues in imiglucerase (2NT1, neutral pH; 3GXI, acidic pH) and in N370S at pH 7.1 and 5.4. d, overlay of all Tyr residues in imiglucerase (2NT1, neutral pH; 3GXI, acidic pH) and in N370S at pH 7.1 and 5.4. Color scheme is same as in Fig. 3.

FIGURE 5.

Thermal denaturation curves for imiglucerase and N370S GCase as measured by DSC. Excess molar heat capacity (kcal mol⁻¹ °C⁻¹) plotted against temperature (°C) from 35 to 70 °C for 16 μm samples in 0.1 m citrate/phosphate buffer, imiglucerase, pH 5.4 (solid line), rGCR, pH 7.1 (dashed line), N370S, pH 5.4 (dash dot dot line) and N370S, pH 7.1 (dotted line).

FIGURE 6.

Stabilization of imiglucerase and N370S GCase by small molecule inhibitor NN-DNJ. NR8383 rat lung alveolar macrophages were incubated with imiglucerase or N370S for uptake. Following the GCase uptake, cells were treated with or without NN-DNJ and lysed at a time course. a, experiment in which the imiglucerase activities were measured with and without 10 μm NN-DNJ treatment. Percentage of enzyme activity remaining was measured for uptake of imiglucerase, subtracted by endogenous GCase activity. Inset, Western blot used for quantification of the protein level remained. The total area of the intact protein band at time 0 on the Western blot was set as 100%. b, Western blot used to quantitate the effect of NN-DNJ on the stabilization of uptaken imiglucerase and N370S GCase. c, percentage of intact protein remained was plotted against the time post uptake. The endogenous rat GCase was not detectable with the polyclonal antibody used.